Evaluation of an enzyme linked immunosorbent assay (ELISA) for determination of insulin in dogs

Between 1970 and 1983, 345 patients with ovarian cancer clinical stage I, II, and III were irradiated postoperatively. Five-year NED survival was achieved in 41.7% of patients. The most important prognostic factors were histological grade and clinical stage of cancer. Postoperative external beam radiotherapy appeared to be highly efficient for the patients with microscopic residual disease, giving 70% 5-year survival, and moderately efficient for patients with small, i.e. < or = 3 cm in diameter residual disease, giving 40% 5-year survival. The optimal technique of irradiation appeared to be the irradiation given to the entire abdominal cavity with additional irradiation coned down to the pelvis. External beam radiotherapy was ineffective in patients with gross residual disease, i.e. > 3 cm in diameter, and useless as palliative treatment given to patients with inoperable cancer of the ovary.     

Enzyme-linked immunosorbent assay for use with nuclear protein-DNA complex antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for identification and quantification of nuclear antigens (nonhistone protein-DNA complexes from chromatin). Until now, the complement fixation assay has been the only immunoassay routinely applied to nonhistone protein-DNA complexes. The ELISA is considerably more sensitive than the micro-complement fixation test for assaying the immunospecificity of nuclear protein-DNA complexes. Dilutions of rabbit antisera as great as 1:6400 could be used to detect nanogram quantities of antigen, chicken reticulocyte chromatin or dehistonized rat liver chromatin.     

Enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone releasing hormone (LH-RH) using a heterobifunctional cross-linking agent, N-[beta-(4-diazophenyl)ethyl]maleimide

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and beta-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and LH-RH4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.     

Enzyme-linked immunosorbent assay for TA-2005-glucuronide in human plasma

Although White Carneau (WC) pigeons are known to be more susceptible to atherosclerosis than Show Racer (SR) pigeons, the reasons for this difference are not fully understood. While no major differences are known in the lipoprotein composition, a difference in the cholesteryl ester (CE) composition was reported. However, there is little information on the activity or specificity of lecithin:cholesterol acyltransferase (LCAT), the major source of plasma CE. In order to determine whether the esterification of cholesterol or other functions of LCAT are compromised in WC pigeons, we studied the various reactions catalyzed by LCAT in the two groups. The cholesterol esterification was found to be significantly lower in WC pigeons, whether assayed with exogenous or endogenous substrates. Furthermore, lyso phosphatidylcholine (PC) esterification and oxidized PC hydrolysis, two other reactions carried out by LCAT, were also lower in WC. We found evidence for the presence of an active lysophospholipase in pigeon plasma, and this activity was also lower in WC compared to SR. A significant increase in the FC/PC ratio, another reported atherogenic risk factor, was found in WC. plasma. Because of the absence of other hydrolytic enzymes in pigeon plasma, LCAT may play an important role in the metabolism of oxidized PC generated during lipoprotein oxidation, and therefore a decrease in its activity in White Carneau pigeons may contribute to increased risk of atherosclerosis.     

An enzyme-linked immunosorbent assay (ELISA) for monitoring guineapigs and rabbits for Bordetella bronchiseptica antibodies

An enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies specific to Bordetella bronchiseptica in guineapigs and rabbits was developed. In conventional and SPF colonies of guineapigs and rabbits, the ELISA was equally successful in detecting infected animals when compared to selective cultivation from the respiratory tract. The ELISA showed a sensitivity of 100% and a specificity of 90% in guineapigs. In rabbits the sensitivity and specificity of the ELISA were 97% and 91% respectively. In rabbit sera from infected colonies, ELISA activity showed a statistically significant correlation with titres obtained in the micro-agglutination test. Since serologically unrelated strains of the bacterium exist, the monitoring of animals for B. bronchiseptica infection by ELISA should be performed with various antigens.     

Use of a multiwell assembly for chemotaxis and evaluation by enzyme-linked immunosorbent assay (ELISA)

A multiwell chamber assembly for chemotaxis tests was designed, which integrates the established microtiter system. A microtiter plate is covered with a plastic plate containing up to 96 holes of the diameter of the microtiter wells. Between the plates, a Nucleopore filter sheet (5 micron) and a silicon rubber gasket is placed. As a model system, human monocytes and lymphocyte-derived chemotactic factors were used. As it was observed that monocytes migrate through the membrane and settle on the bottom of the microtiter wells, an ELISA was adapted for quantitation of cells. After washing and incubation with a xenoantiserum against human monocytes, the bound antibody was quantitated using protein-A-conjugated alkaline phosphatase and p-nitrophenyl phosphate as detection system. The plates were read in a multichannel photometer. Cell numbers were determined directly from a calibration curve established before with varying numbers of monocytes. Current experience allows the following conclusions: The chemotaxis test in microtiter plates is simpler, faster and uses less material than conventional Boyden chambers. Evaluation by ELISA is much faster and more accurate than by microscopy.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies

A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species is discussed.     

Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid--or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.     

Enzyme-linked immunosorbent assay quantification of cytokine concentrations in human meningiomas

OBJECTIVE: To gain insight into the network of cytokine gene expression in the brain tumor microenvironment, we investigated the presence of the following cytokines in freshly excised brain tumors: interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. METHODS: Tumor specimens from nine meningiomas were grown as tissue explants. The supernatants from the explants were tested for the presence of the aforementioned cytokines via the enzyme-linked immunosorbent assay method. RESULTS: IL-6, which is thought to stimulate acute protein phase synthesis, neovascularization, and cell proliferation, was found in all of the samples in greater concentrations than the other cytokines tested. IL-1 beta, another stimulatory cytokine thought to be involved in acute protein phase synthesis and cell proliferation, was also found in 100% of the samples tested, in concentrations significantly lower than those of IL-6. As expected, the presence of IL-2 and IL-4 was not detectable in any of the samples. CONCLUSION: This study is the first to clearly determine the relative concentrations of IL-1 beta and IL-6, using enzyme-linked immunosorbent assay quantification. These findings are an important precursor to future studies using antibodies to IL-1 beta and IL-6 and antibodies to IL-6 receptors to modulate neoplastic growth both in vitro and in vivo.     

Enzyme-linked immunosorbent assay for the detection of Cryptosporidium parvum IgG in the serum of cats

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium parvum IgG in the serum of cats. The ELISA was an indirect ELISA using soluble C. parvum oocyst antigens and a peroxidase-labeled anti-feline IgG secondary antibody. Sera from cats with Toxocara felis, Giardia spp., Aelurostrongylus abstrusus, Isospora felis, Isospora rivolta, Toxoplasma gondii, or Taenia spp. infections were assayed in specificity studies. Following optimization, the ELISA and fecal examination for oocysts were performed on samples from 170 client-owned or humane society source cats and 1 cat inoculated orally with C. parvum oocysts. Cryptosporidium parvum oocysts were detected in feces (4/170; 2.4%), and C. parvum IgG was detected in serum (26/170; 15.3%) from naturally exposed cats. The seroprevalence data suggest that some cats in the geographical area studied were exposed to C. parvum, but persistent oocyst shedding was less common. The ELISA is not useful for predicting oocyst shedding in individual cats.     

Identification and quantitation of sickle cell hemoglobin with an enzyme-linked immunosorbent assay (ELISA)

The experimental details of ELISA for the identification and quantitation of Hb S are presented; the assay is based upon the passive adsorption of Hb S top a solid phase (polystyrene tubes) and the addition of monospecific rabbit antibodies capable of recognizing the (beta 6 Glu leads to Val) substitution in Hb S. After the addition of alkaline phosphatase-conjugated goat antibody to rabbit IgG and substrate, the yellow color produced by hydrolysis of substrate is measured spectrophotometrically. For the identification and quantitation of Hb S in unknown samples, the hemolysate is added to the Hb S-coated tubes before the addition of antibody to Hb S, thus causing an inhibition of the antigen-antibody reaction as evidenced by an absence or reduction of color formation. With this procedure, there is no cross-reactivity with normal hemoglobins, and the immunoassay has a sensitivity in detecting 50 ng quantities of the abnormal hemoglobin in a 5 microgram hemolysate. The assay can be performed on multiple samples in 1 day and offers many advantages over other techniques currently used for the identification and quantitation of Hb S and other abnormal hemoglobins in the clinical laboratory.     

An enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora sp. infection in cattle

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed and optimized for detection of antibodies to Neospora sp. in cattle. Sonicated tachyzoites of Neospora sp. isolated from an aborted bovine fetus were used as antigen. Variability in immunoblot patterns among positive sera, and the fact that all life stages of the parasites are unknown, justified use of a multiple-antigen ELISA to allow for maximum sensitivity. Immunoblot analysis revealed negligible cross-reactions between Toxoplasma gondii antigen and Neospora sp. antisera and between Neospora sp. antigen and antisera from various apicomplexan parasites. The maximum positive-to-negative Vmax (average maximum slope of the optical density over time) ratio was obtained using 200 ng/well of sonicated tachyzoite antigen and a 1:200 serum dilution. Using logistic regression to determine the optimal cutoff point between known infected and noninfected cattle, a sample-to-positive control Vmax ratio of 0.45 was found to maximize the percent correct classification, with an estimated sensitivity of 88.6% and specificity of 96.5%. Use of Neospora caninum antigen following the same protocol demonstrated no difference in ELISA interpretation. Comparison with an existing indirect immunofluorescent antibody (IFA) test showed the ELISA to be the more sensitive and specific test for serodiagnosis of Neospora infection in cattle.     

Enzyme-linked immunosorbent assay of neocarzinostatin chromophore (NCS-chr) by use of a monoclonal antibody against NCS-chr analog

Allelic exclusion at the IgH locus was examined in B lineage cells of wild-type mice and mice unable to express the surrogate light chain molecule lambda 5 using a single-cell PCR approach. By analyzing B precursor cells containing two VHDHJH rearrangements, we found that in wild-type animals, cells are allelically excluded as soon as mu chains are expressed. Furthermore, we provide evidence that in cells expressing D mu proteins VH-->DHJH rearrangement is inhibited. In contrast, in the absence of lambda 5 protein, B precursor cells were allelically "included", indicating that allelic exclusion at the IgH locus requires expression of the pre-B cell receptor either containing a mu chain or a D mu chain. However, although mu chain double-producing B precursor cells are generated in lambda 5-deficient mice, such cells were not detected among surface immunoglobulin positive B cells.     

Comparison of a radioallergosorbent (RAST) inhibition method and a monoclonal enzyme linked immunosorbent assay (ELISA) for aeroallergen measurement

BACKGROUND: Mouse and rat urinary proteins are potent occupational allergens for exposed personnel. Methods of measuring airborne allergens differ greatly, and reported levels of allergens vary considerably between laboratories. OBJECTIVES: To compare the values obtained using two different methods of allergen detection. METHODS: Air samples were collected in rat rooms in Sweden and the United Kingdom at 2 L/min on to polytetrafluoroethylene (PTFE) filters and extracted in buffer containing 0.5% v/v Tween 20. Airborne rat urinary allergen (RUA) was measured in all samples by both RAST inhibition using a polyclonal human serum pool (UK) and a two monoclonal antibody sandwich ELISA employing antibodies specific for Rat n 1.02 (alpha2u-globulin) (Sweden). RESULTS: The two methods gave values which were correlated (r2 log values = 0.72, P<0.0001), but differed by several orders of magnitude (median [range] ratio of RAST inhibition/ELISA = 316 [7-26(80)]. There was a systematic bias: as the absolute values increased, the difference in the measurements increased. The rat urine standards used were antigenically similar. CONCLUSIONS: A large contrast in RUA values obtained from the two assays was observed in this study. This may be primarily due to methodological differences, but variations in antibody specificities or composition of allergenic epitopes in the air samples may contribute. The results demonstrate that standardization of methods and antibodies is necessary before interlaboratory comparisons can be made.     

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

Approximately 0.2% (n = 4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive. The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n = 4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were < or = 3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence = 12%; n = 296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.     

Simplified purification of Listeria monocytogenes listeriolysin O and preliminary application in the enzyme-linked immunosorbent assay (ELISA)

A simplified procedure was developed for purification of listeriolysin O (LLO) of Listeria monocytogenes, consisting of hydroxylapatite adsorption chromatography followed by Sepharose S ion exchange chromatography. The LLO (58 kDa) appeared pure in terms of sodium dodecylsulfate polyacrylamide electrophoresis and immunoblots with polyclonal rabbit immune sera. The purified LLO could be stored at -65 degrees C for 1 year without loss of immunoreactivity. Similarly, flat-bottom microtiter strips from two vendors that had been charged with LLO, could be stored at -65 degrees C for up to 3 months without loss of LLO. Three patients with documented listeriosis developed elevated IgG titres against LLO; 2 of the patients revealed minimally raised IgM titres, as determined with the enzyme-linked immunosorbent assay.     

Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs

Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE.